Sphingosine-1-phosphate (S1P) has been implicated recently in the physiology and pathology of the cardiovascular system including regulation of vascular tone. Pilot experiments showed that the vasoconstrictor effect of S1P was enhanced markedly in the presence of phenylephrine (PE). Based on this observation, we hypothesized that S1P might modulate α₁-adrenergic vasoactivity. In murine aortas, a 20-minute exposure to S1P but not to its vehicle increased the E_max and decreased the EC₅₀ of PE-induced contractions indicating a hyperreactivity to α₁-adrenergic stimulation. The potentiating effect of S1P disappeared in S1P2 but not in S1P3 receptor-deficient vessels. In addition, smooth muscle specific conditional deletion of G_12/13 proteins or pharmacological inhibition of the Rho-associated protein kinase (ROCK) by Y-27632 or fasudil abolished the effect of S1P on α₁-adrenergic vasoconstriction. Unexpectedly, PE-induced contractions remained enhanced markedly as late as three hours after S1P-exposure in wild-type (WT) and S1P3 KO but not in S1P2 KO vessels. In conclusion, the S1P–S1P2–G_12/13–ROCK signaling pathway appears to have a major influence on α₁-adrenergic vasoactivity. This cooperativity might lead to sustained vasoconstriction when increased sympathetic tone is accompanied by increased S1P production as it occurs during acute coronary syndrome and stroke.

Numerous literary data indicate that dynorphin A (DYN-A) has a significant impact on cerebral circulation, especially under pathophysiological conditions, but its potential direct influence on the tone of cerebral vessels is obscure. The aim of the present study was threefold: 1) to clarify if DYN-A is present in cerebral vessels, 2) to determine if it exerts any direct effect on cerebrovascular tone, and if so, 3) to analyze the role of κ-opiate receptors in mediating the effect. Immunohistochemical analysis revealed the expression of DYN-A in perivascular nerves of rat pial arteries as well as in both rat and human intraparenchymal vessels of the cerebral cortex. In isolated rat basilar and middle cerebral arteries (BAs and MCAs) DYN-A (1–13) and DYN-A (1–17) but not DYN-A (1–8) or dynorphin B (DYN-B) induced strong vasoconstriction in micromolar concentrations. The maximal effects, compared to a reference contraction induced by 124 mM K⁺, were 115±6% and 104±10% in BAs and 113±3% and 125±9% in MCAs for 10 µM of DYN-A (1–13) and DYN-A (1–17), respectively. The vasoconstrictor effects of DYN-A (1–13) could be inhibited but not abolished by both the κ-opiate receptor antagonist nor-Binaltorphimine dihydrochloride (NORBI) and blockade of G_i/o-protein mediated signaling by pertussis toxin. Finally, des-Tyr¹ DYN-A (2–13), which reportedly fails to activate κ-opiate receptors, induced vasoconstriction of 45±11% in BAs and 50±5% in MCAs at 10 µM, which effects were resistant to NORBI. DYN-A is present in rat and human cerebral perivascular nerves and induces sustained contraction of rat cerebral arteries. This vasoconstrictor effect is only partly mediated by κ-opiate receptors and heterotrimeric G_i/o-proteins. To our knowledge our present findings are the first to indicate that DYN-A has a direct cerebral vasoconstrictor effect and that a dynorphin-induced vascular action may be, at least in part, independent of κ-opiate receptors.

Introduction: Several recent reports indicate a marked crosstalk between innate immunity and vasoregulation. Anaphylatoxins C3a and C5a have been reported to induce vasoactive effects, but the mechanism of their actions is obscure. Earlier studies reported increased plasma levels of C3 in cardiovascular diseases, especially pulmonary hypertension. Therefore, complement activation may contribute to changes of the vascular tone and reactivity under pathophysiological conditions. In our present study we investigated the effects of C3a, the activate cleavage product of the most abundant complement protein of the human circulation, on the arterial tone and blood pressure as well as the signaling pathways involved. Materials and methods: Thoracic aortic segments were isolated from adult male C57Bl/6 wild type (WT) and knockout (KO) mice deficient in either thromboxane prostanoid receptor (TP KO) or cyclooxygenase 1 (COX1 KO). Isometric tension changes of vascular segments were measured via myography. We studied the effects of C3a by using C3a (63–77) peptide fragment, a specific C3a receptor agonist. Vascular responses were quantitated as percent of reference contraction induced by 124 mM K ⁺ . In addition, we performed experiments monitoring blood pressure (BP) changes in vivo. In these experiments zymosan (30 mg/kg, i.v.), a direct complement activator was administered intravenously, after cannulation of carotid artery and jugular vein in anesthetized (pentobarbital, 90 mg/kg i.p.) mice. Blood samples were taken from other groups of WT mice and plasma TXB ₂ levels were measured 5 minutes after treatment with saline or zymosan. Results: C3a (63–77) evoked a pronounced vasoconstriction in WT vessels (33±5%), which effect remained unaltered in the absence of endothelium (37±7%). The vessels of COX1 KO mice showed a decreased response (10±2%), and the C3a fragment caused a similarly reduced constriction in TP KO vessels (4±1%). In our in vivo experiments the administration of zymosan led to an increase of BP in WT mice (with an average of 14±5 mmHg), whereas causing a drop of 22±3 mmHg in TP KO mice's BP. In accordance, zymosan caused a two‐fold increase in the plasma level of thromboxane B ₂ in WT animals. Conclusions: Our experiments showed, that C3a (63–77) causes vasoconstriction in a non‐endothelium‐dependent manner, and this effect is mediated by COX1 and TP. These results propose that C3a‐mediated vasoconstriction is mediated by smooth muscle derived thromboxane A ₂ . In addition, C3a (63–77) induce hypertension, which is also mediated by thromboxane A ₂ . These results altogether indicate that C3a is involved in the regulation of vascular tone and/or reactivity via stimulating thromboxane A ₂ release. Support or Funding Information Grant support: Semmelweis Research and Innovation Fund (STIA‐M‐17); Ministry of Human Capacities of Hungary (ÚNKP‐18‐2‐I‐SE‐12, FIKP‐2018 and EFOP‐3.6.3‐VEKOP‐16‐2017‐00009) and the Hungarian NRDIO (OTKA K‐115623, K‐125174 and NVKP_16‐1‐2016‐0042) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Tachykinins (TKs) are involved in both the physiological regulation of urinary bladder functions and development of overactive bladder syndrome. The aim of the present study was to investigate the signal transduction pathways of TKs in the detrusor muscle to provide potential pharmacological targets for the treatment of bladder dysfunctions related to enhanced TK production. Contraction force, intracellular Ca²⁺concentration, and RhoA activity were measured in the mouse urinary bladder smooth muscle (UBSM). TKs and the NK2 receptor (NK2R)-specific agonist [β-Ala⁸]-NKA(4–10) evoked contraction, which was inhibited by the NKR2 antagonist MEN10376. In Gα_q/11-deficient mice, [β-Ala⁸]-NKA(4–10)-induced contraction and the intracellular Ca²⁺concentration increase were abolished. Although G_q/11proteins are linked principally to phospholipase Cβ and inositol trisphosphate-mediated Ca²⁺release from intracellular stores, we found that phospholipase Cβ inhibition and sarcoplasmic reticulum Ca²⁺depletion failed to have any effect on contraction induced by [β-Ala⁸]-NKA(4–10). In contrast, lack of extracellular Ca²⁺or blockade of voltage-dependent Ca²⁺channels (VDCCs) suppressed contraction. Furthermore, [β-Ala⁸]-NKA(4–10) increased RhoA activity in the UBSM in a G_q/11-dependent manner and inhibition of Rho kinase with Y-27632 decreased contraction force, whereas the combination of Y-27632 with either VDCC blockade or depletion of extracellular Ca²⁺resulted in complete inhibition of [β-Ala⁸]-NKA(4–10)-induced contractions. In summary, our results indicate that NK2Rs are linked exclusively to G_q/11proteins in the UBSM and that the intracellular signaling involves the simultaneous activation of VDCC and the RhoA-Rho kinase pathway. These findings may help to identify potential therapeutic targets of bladder dysfunctions related to upregulation of TKs.

Muscarinic acetylcholine receptors are of utmost importance in physiological regulation of micturition and also in the development of voiding disorders. We demonstrate that the RhoA-Rho-associated kinase (ROCK) pathway plays a crucial role in contractions induced by cholinergic stimulation in detrusor muscle. Activation of RhoA is mediated by both M₂ and M₃ receptors as well as by G_i but not G_q/11 proteins. The G_i-RhoA-ROCK pathway may provide a novel therapeutic target for overactive voiding disorders.

We examined the vasoactive effect of estradiol in a rat model of early PCOS and the influence of vitamin D deficiency (VDD). We created a model of chronic hyperandrogenism and VDD in adolescent female Wistar rats (N = 46) with four experimental groups: vitamin D supplemented (T-D+), VDD (T-D-), hyperandrogenic and vitamin D supplemented (T+D+), and hyperandrogenic and VDD (T+D-). T+ groups received an 8-week-long transdermal Androgel treatment, D-animals were on vitamin D-reduced diet and D+ rats were supplemented orally with vitamin D3. Estrogen-induced vasorelaxation of thoracic aorta segments were measured with a wire myograph system with or without the inhibition of endothelial nitric oxide synthase (eNOS) or cyclooxygenase-2 (COX-2). The distribution of estrogen receptor (ER), eNOS and COX-2 in the aortic wall was assessed by immunohistochemistry. VDD aortas showed significantly lower estradiol-induced relaxation independently of androgenic status that was further decreased by COX-2 inhibition. COX-2 inhibition failed to alter vessel function in D+ rats. Inhibition of eNOS abolished the estradiol-induced relaxation in all groups. Changes in vascular function in VDD were accompanied by significantly decreased ER and eNOS staining. Short-term chronic hyperandrogenism failed to, but VDD induced vascular dysfunction, compromised estrogen-dependent vasodilatation and changes in ER and eNOS immunostaining.